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2013

Detection and characterization of antibioticresistance plasmids in Cheney biosolidsGaayathiri ParamasivamEastern Washington University

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Recommended CitationParamasivam, Gaayathiri, "Detection and characterization of antibiotic resistance plasmids in Cheney biosolids" (2013). EWU MastersThesis Collection. 241.http://dc.ewu.edu/theses/241

DETECTION AND CHARACTERIZATION OF ANTIBIOTIC RESISTANCE PLASMIDS IN

CHENEY BIOSOLIDS

A Thesis

Presented To

Eastern Washington University

Cheney, Washington

In Partial Fulfillment of the Requirements

For the Degree

Biology (Master of Science)

By

Gaayathiri Paramasivam

Winter 2013

ii

THESIS OF GAAYATHIRI PARAMASIVAM APPROVED BY

DATE

DR. PRAKASH BHUTA, GRADUATE STUDY COMMITTEE

DATE

DR. ANDREA CASTILLO, GRADUATE STUDY COMMITTEE

DATE

DR. LAVONA REEVES, GRADUATE STUDY COMMITTEE

iii

MASTER’S THESIS

In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Eastern Washington University, I agree that the JFK Library shall make copies freely available for inspection. I further agree that copying of this project in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without my written permission.

Signature Date

iv

ABSTRACT

Biosolids are organic matter produced at the end of sewage treatment process. It has

been shown that during sewage treatment, resistant bacteria are selected because of

the presence of antibiotics and their byproducts. These resistant bacteria are more likely

to transfer resistance genes to other bacteria. In the current study, we examined

Cheney biosolids for the presence of drug resistant bacteria and the role of these

bacteria in transfer of resistance genes to others. We screened 100 bacteria for drug

resistance and found that 68% of the isolates were resistant to two or more drugs

tested. Plasmids were separated from the resistant bacteria and 13.2% of them showed

the presence of plasmids. These resistance plasmids were introduced into E. coli MM294

to screen for the presence of antibiotic resistance genes. Plasmids were isolated from

the transformants and 77.7% of the transformants showed the presence of plasmids

with similar size and mobility on an agarose gel. The plasmids extracted from the

transformants were digested with a restriction enzyme, EcoRI to verify the presence of

multiple plasmids in the samples. The resistant bacteria (13.2%) that showed the

presence of plasmid were tested whether they were conjugative or mobilizable type.

Unfortunately, none of the isolates were conjugative or mobilizable plasmid. In short,

Cheney biosolids do contain drug resistant bacteria, so there is a chance that these

resistant bacteria will transfer their resistance genes to other bacteria present in

biosolids.

v

ACKNOWLEDGEMENTS

I would like to thank Mike Lambert at the Cheney wastewater plant for his help in collecting biosolid samples. I would like to thank Dr. Steve Moseley at University of Washington for coliphage samples and bacterial cultures. I would also like to thank Dr. Nicholas Burgis at EWU Chemistry Department for bacterial cultures. I would like to thank Dr. Prakash Bhuta, Dr. Andrea Castillo and Dr. LaVona Reeves for their help and guidance. Finally, I would like to thank my friends and family for their support and much more.

vi

TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………………………………………iv

ACKNOWLEDGEMENTS…………………………………………………………………………………....v

1.0 INTRODUCTION……………………………………………………………………………………...…1 1.1 Significance of Antibiotics………………………………………………………...1 1.2 Issues with Drug Resistant Bacteria…………………………………………..4 1.3 Development of Antibiotic Resistance…………………………………...…6 1.4 Plasmids and Antibiotic Resistance……………………………………….….10 1.5 Accumulation of Antibiotics in the Environment…………………..….12 1.6 Production of Biosolids…………………………………………………………....13 1.7 Regulations for Biosolids………………………….…………………………..….16 1.8 Potential Hazards of Biosolids………………………………………………....17 1.9 Significance of Bacteria in Enterobacteriaceae Family………………18 1.10 Plasmid Extraction and Transformation……………………………….19 1.11 Phage Sensitivity Assay………………………………………………………..21 1.12 Mechanism of Conjugation…………………………………………….……24 1.13 Objectives…………………………………………………………………….……..28

2.0 MATERIALS and METHODS…………………………………………….…………………..…..….29

2.1 Bacterial Growth Media…………………………………………………………....29 2.2 Preparation of Antibiotic Stock Solutions………………………………..…29 2.3 Preparation of Buffer and Solutions…………………………………………..30 2.4 Collection of Biosolids and Inoculation onto Selective Media…….31 2.5 Bacterial Strains Used………………………………………………………………..31 2.6 Long Term Storage of Bacterial Isolates……………………………………..33 2.7 Plasmid DNA Extraction……………………………………………………………..33 2.8 Agarose Gel Electrophoresis………………………………………………………34 2.9 Identification of Bacteria……………………………………………………………35 2.10 Preparation of Competent Cells…………………………………………….35 2.11 Transformation……………………………………………………………………..36 2.12 Restriction Digestion……………………………………………………………..37 2.13 Assay of Phage Sensitivity……………………………………………………..37 2.14 Tripartite Mating…………………………………………………………………..38

vii

TABLE OF CONTENTS

3.0 RESULTS and DISCUSSION………………………………………………………………………….…40

3.1 Collection of Biosolids and Isolation of Resistant Bacteria…………….....40 3.2 Isolation of Plasmids from Resistant Bacteria……………………………………47 3.3 Identification of Resistant Bacteria………………………………………………..….50 3.4 Transformation……………………………………………………………………………..….53 3.5 Restriction Digestion……………………………………………………………..………….59 3.6 Phage Sensitivity Testing…………………………………………………………..………64 3.7 Tripartite Mating………………………………………………………………………..…….66

4.0 CONCLUSIONS……………………………………………………………….………………………….….69

5.0 REFERENCES………………………………………………………………………………………….………71

VITA……………………………………………………………………………………………………….…..………79

viii

List of Tables and Figures:

Table 1 – Antibiotics: classes, targets and resistance mechanisms

Table 2 – The U.S EPA – Pathogen concentration limits

Table 3 – Clinical syndromes caused by members of the Enterobacteriaceae family

Table 4a – Bacterial strains used

Table 4b – Phages used

Table 5a – Resistance profile of the isolates X1-X36

Table 5b – Resistance profile of the isolates X37-X68

Table 6 – Identity of resistant bacteria and their antibiotic resistance profile

Table 7 – Transformation efficiency of parental plasmids

Figure 1 – Mechanism of horizontal gene transfer (HGT) in bacteria

Figure 2 – Mechanism of antibiotic resistance

Figure 3 – Production of biosolids

Figure 4 – Mechanism of transformation in bacteria

Figure 5 – Infection of RNA phage and cell lysis

Figure 6 – Mechanism of conjugation in bacteria

Figure 7 – Percent of isolates resistant to different antibiotics

Figure 8 – Percent of isolates resistant to number of drugs used

Figure 9 – Antibiotic sensitivity testing of the isolates

Figure 10a and 10b – Agarose gel showing the presence of plasmids

Figure 11 – API-20E strips used for the identification of bacteria

Figure 12a to 12f – A comparison between plasmids isolated from transformants with parental plasmids

Figure 13a to 13g – Restriction digestion of the plasmids extracted from transformants

Figure 14 – Phage sensitivity testing

Figure 15a – Resistance profile of the donor, helper and recipient strains

Figure 15b – Tripartite mating process

1

1. Introduction:

1.1 Significance of Antibiotics:

Antibiotics are chemicals that restrict the growth of bacteria. They are divided into

two major categories based on their effect on a target cell: bacteriostatic (growth

inhibitor) and bactericidal (killing agent) (Davies, 2010; Walsh, 2003). Antibiotics affect

bacterial growth by many different mechanisms including: 1. disruption of bacterial cell

wall (e.g., Penicillin), 2. inhibition of protein synthesis (e.g., Aminoglycosides and

Tetracyclines), and 3. inhibition of DNA replication and transcription (e.g., Quinolones

and Rifampin). The most commonly used antibiotics and those used in this study are

listed in Table 1. Antibiotics are used everywhere - in agriculture, aquaculture, livestock,

veterinary and human treatment. From the 1950s, antibiotics such as oxytetracycline

and streptomycin have been used as pesticides when growing fruit, vegetable, and

ornamental plants. Streptomycin is primarily used for control of fire blight in apples,

pears and plants of the Rosaceae family. In the USA, approximately 10,000 tons of

antibiotics are used on plants (McManus et al., 2002). Animal farms and aquaculture are

also using antibiotics to promote growth and prevent diseases (Cabello, 2006). For

example, in swine breeding farms, antibiotics are detected in dust from the air

ventilators (Hamscher et al., 2003). It has been shown that antibiotic misuse or overuse

has resulted in the development of drug resistant bacteria in the environment (Rhodes

et al., 2000). Such resistant bacteria pass

2

on their resistance traits to successive generations (vertical transmission) and to other

members of the bacterial community (horizontal gene transfer) (Davies, 2010;

Freifelder, 1987).

3

Table 1: Antibiotics: classes, targets and resistance mechanisms

Class / Generic

name

Common uses Target Resistance Mechanism

Aminoglycosides Kanamycin

Streptomycin

E. coli, Klebsiella, Pseudomonas

infections

Anti-tuberculosis

Binds to 30S subunit of bacterial ribosome, thereby

inhibits protein synthesis

Phosphorylation, acetylation,

nucleotidylation, drug efflux and target

alteration

Penicillin

Ampicillin

Streptococcal infections,

Syphilis, and Lyme disease

Disrupts the peptidoglycan layer

of bacterial cell wall

Hydrolysis, drug efflux and target alteration

Quinolones

Nalidixic acid

Ciprofloxacin

Urinary tract infections,

pneumonia, bacterial

diarrhea, and mycoplasmal

infections

Bind to DNA gyrase A, thereby inhibits

DNA replication and transcription

Gyrase mutations, reduced uptake and

drug efflux

Tetracyclines

Tetracycline

Syphilis, Lyme disease and

mycoplasmal infections

(also used to treat infections

in plants and animals)

Binds to 30S subunit of bacterial ribosome, thereby

inhibits protein synthesis

Monooxygenation, drug efflux and target

alteration

Others

Rifampin

Gram positive and

mycobacteria

Binds to the β subunit of RNA polymerase and

inhibits transcription

ADP-ribosylation, drug efflux and target

alteration

Chloramphenicol (rarely used in the

U.S)

Meningitis, MRSA, topical use, or for low cost internal treatment.

Inhibits bacterial protein synthesis by binding to the

50S subunit of the ribosome

Acetylation, drug efflux and target

alteration

1.2 Issues with Drug Resistant Bacteria:

4

In 1928, Alexander Fleming discovered the first naturally occuring antibiotic,

penicillin. In the 1940s, even before the introduction of penicillin into clinical practice, a

bacterial enzyme penicillinase was discovered. Penicillinase breaks down penicillin by

targeting the β-lactam ring present in its structure (Abraham and Chain, 1940).

Antibiotic producing bacteria and fungi developed natural resistance to protect

themselves from the antibiotics that they produce. This shows the prevalance of

antibiotic resistance in natural settings (Walsh, 2003). However, introduction of

antibiotics in clinical practice has created enormous pressure on bacteria and selected

them for drug resistance. A strong correlation between the use of antibiotics and a

development of resistance has been observed over the past half-century (Shlaes et al.,

1997). This is especially true for β-lactam class of antibiotics and their corresponding

inactivating enzyme, β-lactamases (Damoa-Siakwan, 2005; Shlaes et al., 1997). At this

time, several classes of β-lactamases have been identified, comprising up to 1000

different β-lactamases (Bush and Jacoby, 2010). The use of antibiotics worldwide in

clinical practice has led to multidrug resistant (MDR) and extensively drug resistant

(XDR) strains of bacteria e.g., MDR and XDR Mycobacterium tuberculosis (Gandhi et al.,

2006 and Sekiguchi et al., 2007). The Centers for Disease Control and Prevention (CDC,

2008) estimate the annual cost of treating infections caused by drug resistant bacteria

to be 5.7 to 6.8 billion U.S. dollars. This shows the persistence of drug resistance in

bacterial populations.

The resistance genes that confer antibiotic resistance are prevalent and widespread

among bacterial communities because of horizontal gene transfer (HGT) (Shlaes et al.,

5

1997). The HGT mechanisms include: 1. transformation (cell free DNA uptake), 2.

conjugation (bacterial mating) and 3. transduction (viral infection). These mechanisms

have played a significant role in the evolution and transmission of β-lactam resistance

among bacteria. For instance, Pseudomonas aeruginosa became resistant to β-lactams

and aminoglycosides (another class of antibiotics), thus evolved into a life threating

pathogen. Patients with cystic fibrosis are most susceptible to P. aeruginosa as this

pathogen is highly opportunistic and infect damaged tissues by avoiding human immune

defenses (Curran et al., 2004; Horrevorts et al., 1990).

Similarly, a gram-negative pathogen, Acinetobacter baumnanni is primarily hospital

acquired (nosocomial) causing serious infections and death (Peleg et al., 2008). These

organisms persist in soil and water. Several Acinetobacters are resistant to various

classes of drugs such as β-lactams, aminoglycosides, quinolones and tetracyclines. In

addition, many Acinetobacters are naturally competent for DNA uptake and have high

rates of natural transformation. This supports the idea that these organisms are capable

of exchanging resistance genes with others (Peleg et al., 2008). The toxin producing

anaerobe, Clostridium difficile is also hospital acquired and found to cause severe

intestinal infections. This gram-positive spore former can be readily transmitted by

hospital personnel and equipments (Gifford and Kirkland, 2006). Once ingested, these

organisms reside in the human gut but their numbers are kept low by normal gut flora.

If the gut flora are eliminated by antibiotics, these Clostridia colonize the entire gut and

release toxins which can result in the serious intestinal infection, pseudomembranous

6

colitis. Recently, hypervirulent toxin producing strains have also been recognized

(Vernaz et al., 2009).

The most famous superbug is the gram-positive bacterium, Staphylococcus aureus. It

is a nasal commensal in 30% of the population and causes common skin infections such

as impetigo and boils (Enright et al., 2002 and Tenover et al., 2001). In 1959, the

antibiotic methicillin was discovered to suppress penicillin resistant S. aureus, but

within 3 years methicillin-resistant S. aureus (MRSA) had arisen (Barber, 1961; DeLeo

and Chambers, 2009). The MRSA is a well-known hospital pathogen. More than 10% of

bloodstream infections in hospitals are due to MRSA and patients with MRSA have

worse outcomes than those with methicillin-sensitive S. aureus (Selvey et al., 2000). In

recent years, identification of MRSA in healthy individuals in a community has become

increasingly common (Delaney et al., 2008; Klevens et al., 2007). Thus, an increase in the

development of drug resistance among many opportunistic pathogens presents a major

health and economic concern.

1.3 Development of Antibiotic Resistance:

Bacteria develop antibiotic resistance primarily by: point mutations and horizontal

gene transfer (HGT). The development of resistance by point mutation occurs only by

chance i.e., bacteria develop mutations at a rate faster than other organisms, as they

have shorter generation times and the rapid rate of DNA replication may produce errors

leading to mutations (Walsh, 2003). Some of these mutations give selective advantage

for their survival, such as protection against antibiotics in their surroundings. Such

7

resistance mutation can remain in the bacterial population and it can be passed on to

successive generations through vertical transmission (from parent to offspring) (Walsh,

2003). For instance, M. tuberculosis developed multidrug resistance exclusively by

spontaneous mutation. Streptomycin is the antibiotic most commonly used against M.

tuberculosis but streptomycin resistant strains are creating problems in patients with

compromised immune system and resulting in high morbidity and mortality rates (Shah

et al., 2007; Sotgiu et al., 2009; Velayati et al., 2009). Mutations in the ribosomal protein

S12 or within the 530 loop of 16S rRNA are responsible for streptomycin resistance in M.

tuberculosis. A single amino acid change from lysine to either arginine or threonine on

S12 and 16S rRNA is enough for M. tuberculosis to become resistant to streptomycin

(Finken et al., 1993). Thus, mutation plays an essential role in the development of drug

resistance in bacteria. However, bacteria can also gain drug resistance by other means

such as horizontal gene transfer (Figure 1) (Fajardo, et al., 2009; Freifelder, 1987;

Phornphisutthimas et al., 2007).

There are three types of mechanisms involved in horizontal gene transfer (HGT): 1.

Transformation, 2. Conjugation, and 3. Transduction (Figure 1). Transformation is a

process by which bacteria take a small amount of cell free DNA from the surroundings

which is released from other dead bacteria. Conjugation is a process of gene transfer

that needs direct contact between a donor and a recipient cell. The donor cell carries a

special type of plasmid or fertility (F) factor which promotes gene transfer. Transduction

is a process of gene transfer that occurs when viruses carrying bacterial genes infect

bacteria. These mechanisms are illustrated in Figure 1. The genetic elements that are

8

transferred by HGT can be any of the following: small fragment of bacterial genomic

DNA, a plasmid, transposon, integron and bacteriophages (Freifelder, 1987; Heuer et al.,

2002). Hence, it is important to analyze the presence of drug resistant bacteria and

plasmids in biosolids and also for their role in transferring resistance genes to others in

the bacterial community.

9

Figure 1: Mechanism of horizontal gene transfer (HGT) in bacteria. The three types of mechanisms involved in HGT: 1. Transformation (uptake of cell-free DNA), 2. Conjugation (bacterial mating), and 3. Transduction (gene transfer by viruses)

10

1.4 Plasmids and Antibiotic Resistance:

A plasmid is a circular, autonomously replicating DNA molecule. It may carry genes

that can be beneficial to their host (e.g., genes which code for drug resistance, virulence

factors etc.) (Carattoli, 2009). The resistance genes within the plasmids may be

responsible for inactivation of antibiotics and the mechanisms involved are: 1. drug

modification, 2. drug degradation, 3. drug efflux and 4. drug target alteration (Figure 2)

(Walsh, 2003). The drug modification and degradation processes involve specific

enzymes encoded by the resistance genes carried on plasmids. These enzymes

recognize and bind to the incoming antibiotic molecules and either cleave or modify

them chemically to prevent antibiotics from binding to their targets. Another

mechanism called drug efflux involves efficient transport of antibiotics from the inside

to the outside of the bacterial cell. In the target alteration mechanism, a specific cellular

target for a drug is altered through mutation to prevent the drug from binding and

making them ineffective. These four mechanisms are illustrated in Figure 2. All these

mechanisms can be regulated by the antibiotic resistance genes present on plasmids

(Walsh, 2003).

11

Figure 2: Mechanism of antibiotic resistance. The four mechanisms involved in antibiotic resistance are: 1. drug inactivation, 2. drug modification, 3. drug efflux, and 4. target alteration.

Again, the resistance (R) plasmids can be a threat to humans and animals because it

can be transferred laterally among bacteria of different genera by HGT (Figure 1)

(Couturier et al., 1988). Based on their ability to move between different bacteria,

12

plasmids are categorized into: 1. self-transmissible or conjugative, and 2.

nonconjugative or mobilizable plasmids (Salyers and Amabile-Cuevas, 1997).

Conjugative plasmids carry tra genes needed for conjugation and they do not need any

external support for their gene transfer. By contrast, nonconjugative plasmids lack tra

gene functions, so they depend on the tra genes of other conjugative plasmids for their

gene transfer (Freifelder, 1987). For instance, when planktonic bacteria carrying

plasmids come into contact with a biofilm population composed of recipient cells with

no plasmids, planktonic bacteria will form biofilms and transfer infectious plasmids to

the recipient. Plasmids can transfer antibiotic resistance and virulence factors, threofore

plasmids involved in biofilm formation can produce infecions that are hard to treat

(Ghigo, 2001). Since, R plasmids play a significant role in gene exchange between

organisms, it is important to analyze the presence of R plasmids in environmental

samples such as biosolids.

1.5 Accumulation of Antibiotics in the Environment:

As antibiotics are poorly absorbed in the gut of animals and humans, a large amount

of them are excreted unchanged in feces and urine (Sarmah et al., 2006). For example,

40-90% of sulphonamides and tetracyclines are excreted unchanged in feces after

passage through the gastro-intestinal tract of swines (Winckler and Grafe, 2001).

Antibiotics are not easily degraded in soils, especially when they are spread onto soils as

contaminants in sludge (Halling-Sorensen, 1998; Thiele-Bruhn, 2003). They can persist in

the environment for a long time (2-6 months) and can reach ground water or aquatic

13

sediments through application of liquid manure or sewage sludge as fertilizers (Kuhne et

al., 2001; Kummerer, 2001; Richardson and Bowron, 1985). In the USA, a nationwide

survey was conducted regarding the presence of pharmaceuticals in river water

samples. The data revealed a number of veterinary and human antibiotics in

concentrations of 0.7% in 27% of 139 river water samples (Kolpin et al., 2002).

Application of dairy farm manure to a garden soil caused a 70% increase in bacterial

resistance against ampicillin, penicillin, tetracycline, vancomycin and streptomycin

(Esiobu et al., 2002). Therefore, application of manure as a fertilizer increases the

chance of drug resistance among bacterial communities (Sarmah et al., 2006).

1.6 Production of Biosolids:

Biosolids are nutrient-rich organic materials produced during treatment of

wastewater. Once wastewater from households and industry reach a sewage treatment

plant, it undergoes several physical, chemical and biological processes to reduce organic

matter, eliminate heavy metals and disease causing pathogens. First, the raw sewage is

filtered to remove large solid materials and the liquid undergoes aerobic and anaerobic

digestion. In the aerobic treatment process, wastewater is mechanically agitated with

the supply of air and the temperature is maintained at 15-20°C for 40 days to activate

aerobic microorganisms which reduce biological oxygen demand and stabilize

suspended solids in the wastewater (U.S. EPA, 1994). This is followed by an anaerobic

digestion process in which the temperature is maintained at 20-55°C for 40 days in the

absence of oxygen to transform organic materials in the sludge to gases such as

methane and carbon dioxide. By the end of aerobic and anaerobic digestion processes,

14

there will be a reduction in the quantity of solids and pathogenic organisms. The sludge

is then air dried for 2 months and lime is added to raise the pH and eliminate odors (U.S.

EPA, 1994). Later, composting materials like saw dust and yard waste are added to the

sludge and the final product is called biosolids. The processes involved in the production

of biosolids are illustrated in Figure 3.

The biosolids are recycled and applied as fertilizer to promote plant growth as they

are rich in nutrients like nitrogen, phosphorous, potassium and trace amounts of

calcium, copper, iron, magnesium, manganese, sulfur and zinc (U.S. EPA, 2001). In the

United States, approximately 5.6 million dry tons of biosolids are generated annually

and 60% of them are used as fertilizer whereas the rest is incinerated or dumped in

landfills (National Research Council, 2002). Biosolids are used as a fertilizer in public

parks, agricultural lands, forest lands, reclamation sites, golf courses, road sides, plant

nurseries, lawns, and home gardens (U.S. EPA, 1994).

15

Figure 3: Production of biosolids. The biosolids undergo several processes before they are applied to lands. These include 1. aerobic and anaerobic digestion, 2. lime stabilization, and 3. composting.

Raw sewage

Filtered to remove solid material

Liquid is subjected to aerobic and anaerobic digestion

Liquid is chlorinated

Released into environment

Suspended solid material is removed

Stabilized using lime and composting materials: wood chips,

yard waste

Biosolids

16

1.7 Regulations for Biosolids:

As biosolids are being applied to lands as a fertilizer, it must meet some regulations

and quality standards before its application. The U.S Environmental Protection Agency

(EPA) established a set criterion for the concentration of heavy metals and pathogens

(e.g., bacteria, viruses and parasites) permitted in biosolids for the safety of humans and

animals. The EPA classified biosolids into two types based on their pathogen content -

class ‘A’ and class ‘B’ (EPA, 1994). The class ‘A’ type undergoes some additional steps

like heat drying, pasteurization, β-ray irradiation and gamma ray irradiation to further

reduce the number of pathogens - but those treatment methods vary among treatment

plants. In general, class ‘A’ is tested for the following indicator organisms and

pathogens: Salmonella, fecal coliforms, enteric viruses and helminthes ova, whereas

class ‘B’ is tested only for fecal coliforms such as E.coli, Citrobacter, Enterobacter and

Klebsiella. The number of pathogens and their concentration allowed in class ‘A’ and ‘B’

are shown in Table 2. The class ‘A’ type is recommended by the U.S EPA for application

to home gardens and public parks, and the class ‘B’ for crop lands and forestry. The

potential risks associated with application of biosolids are discussed in the next section.

17

Table 2: The U.S EPA - Pathogen Concentration Limits

Pathogen / indicator and class Standard concentration limits (dry wt.)

Class A Salmonella Fecal coliforms Enteric viruses Viable helminthes ova

< 3 MPN/4 g total solids < 1000 MPN/g total solids < 1 PFU/4g total solids < 1 PFU/4g total solids

Class B Fecal coliform density

< 2 million MPN/g total solids

Note: MPN – most probable numbers; PFU – plaque forming unit. Source: The U.S. EPA, 2001

1.8 Potential Hazards of Biosolids:

Although the EPA regulates biosolids use, there are risks associated with such

application. Studies show that biosolids are unsafe for land application due to: 1.

dispersion of bioaerosols from application sites (Baerisch et al., 2007; Pillai et al., 1996),

2. contamination of food and groundwater (Edmonds, 1976; Sidhu and Toze, 2009;

Tamminga et al., 1978; Wei et al., 2010), and 3. prevalence of viruses and resistant

strains of bacteria (Binh et al., 2008; Ewert and Paynter, 1980; Ward et al., 1981; Wong

et al., 2010). It has been shown that gene transfer occurs between different strains of

Enterococcus faecalis under natural conditions in wastewater treatment plants

(Marcinek et al., 1998). Several pharmaceutical compounds (ibuprofen, acetaminophen,

gemfibrozil), and antibiotics (ciprofloxacin, ofloxacin, azithromycin) persist in biosolids

despite of all the treatment processes (Radjenovic et al., 2009; Spongberg and Witter,

2008). Presence of antibiotics in biosolids put selective pressure on bacteria and select

18

for drug resistant determinants. These resistant organisms can act as a reservoir for

drug resistance plasmids (Binh et al., 2008; Viau and Peccia, 2009). In addition, human

and bacterial viruses present in sewage can be maintained because they are more

resistant to ammonia and heat treatments (Burge et al., 1983; Lund et al., 1996; Viau

and Peccia, 2009). These studies support an idea that resistant bacteria transfer

resistance genes to other organisms through vertical and horizontal transmission.

Therefore, it is necessary to examine these possibilities in biosolids before their land

application.

1.9 Significance of Bacteria in Enterobacteriaceae Family:

The Enterobacteriaceae is a large family of gram negative bacteria and some

members are part of a normal gut flora of humans and animals. However, members of

this family such as E.coli, Salmonella and Proteus are known pathogens while others like

Enterobacter, Klebsiella and Serratia cause secondary wound infections, respiratory and

urinary tract infections (Table 3). As these members are found in human and animal

intestine, they are present in sewage in large quantity and in biosolids in spite of the

wastewater treatment processes. It has been shown that members of

Enterobacteriaceae are resistant to multiple drugs and infections caused by these

resistant organisms are hard to treat (Paterson, 2006; Salyers and Amabile-Cuevas,

1997). Studies show that members of this family (e.g., Salmonella and other fecal

coliforms) are able to regrow and colonize after biosolids are applied to lands (Zaleski et

al., 2005). As members of the Enterobacteriaceae family are enteric pathogens, it is

crucial to study these organisms in biosolids.

19

Table 3: Clinical syndromes caused by members of the Enterobacteriaceae family

Bacteria Habitat / Source

Disease

Citrobacter spp. Soil, water and wastewater Urinary tract infection, infant meningitis and sepsis

Enterobacter spp. Soil, water and human intestine

Urinary and respiratory tract infections

E.coli 0157:H7 Human intestine Diarrhea and severe kidney failure

Klebsiella pneumonia Nosocomial; Spread by hospital personnel and

equipment

Pneumonia, diarrhea bacteremia and sepsis

Proteus spp. Soil, water, wastewater and human intestine

Wound infections, pneumonia and septicemia

Serratia marcescens Nosocomial; Spread by hospital personnel and

equipment

Urinary tract, wound infection and septicemia

Salmonella spp. Shigella spp.

Contaminated food and water, animal and human

intestine

Diarrhea, enteric fever, septicemia and reactive arthritis

1.10 Plasmid Extraction and Transformation:

The presence of resistance genes on plasmids can be examined by transformation. For

such an analysis, plasmids can be extracted from antibiotic resistant bacteria and these

cell-free plasmids can be mixed with an antibiotic sensitive laboratory strains of E. coli.

20

Through the transformation process, bacteria take a small amount of cell free DNA from

their surroundings and keep them in their cytoplasm as a plasmid or incorporate them

into their chromosome (Andersson and Hughes, 2010). The bacteria that are susceptible

to transformation in both natural and artificial environments are called competent cells.

In a lab, competence can be achieved by treating the cells with chemicals (e.g., CaCl2)

and using high voltage electric pulse (electroporation) (Froger and Hall, 2007; Hill et al.,

1992). The process of transformation is illustrated in Figure 4. Using transformation

process, drug resistance genes (R) present on plasmids can be identified.

Figure 4: Mechanism of transformation in bacteria. Transformation is a process by which bacteria take a small amount of cell free DNA from their surroundings. Once the DNA enters a

21

cell, it will be maintained stably as a plasmid or may be integrated into their chromosome. In either case, the new DNA is passed on to successive generations.

1.11 Phage Sensitivity Assay:

To determine whether plasmids are of the conjugative or non-conjugative type, a

phage sensitivity assay can be used. Viruses can replicate only in specific host. Based on

the host they live in, they are classified into three types: 1. viruses that infect bacteria

are called bacteriophages or phages, 2. viruses that infect plants are referred as plant

viruses, and 3. viruses that infect animals and humans. For the identification of

conjugative and non-conjugative plasmids, bacteriophages or phages can be used.

Bacteriophages basically consist of a nucleic acid molecule (DNA or RNA) surrounded

by a protein coat called capsid (Lipton and Weissbach, 1969). After infection, phages use

the bacterial ribosomes, protein-synthesizing factors and energy-generating systems for

their replication, hence phages can only multiply in metabolizing bacterial cells (Sobsey

et al., 1995). Phages that infect E.coli bacteria are called coliphages. Attachment or

receptor sites for coliphages are located on different parts of bacteria. Based on the

receptor sites, coliphages are classified into two types: somatic and male-specific (F+)

coliphages. Some receptors for coliphages are located on the bacterial cell wall and are

present all the time. These receptors are recognized by phages known as somatic

phages. This implies that somatic phages can infect host bacteria at any time, and these

phages will attach to even dead bacteria. Some examples of somatic phages are T4, T5,

λ and P22 phages (Furuse et al., 1981; Long et al., 2005). Receptor sites for other

22

coliphages are located on the fertility (sex) fimbriae of host bacteria. These fimbriae are

produced only by bacteria carrying F-factor in the log growth phase under optimal

conditions. Coliphages which utilize these receptor sites are known as male-specific (F+)

phages. Some examples of male-specific phages are Qβ, M13, MS2 and f1 (Furuse et al.,

1981; Long et al., 2005; Sobsey et al., 1995).

Bacteriophages share many fundamental properties with human viruses. For

example, F+ RNA coliphages and polioviruses, both have an icosahedral capsid with a

diameter of about 25nm and a single stranded (ss) RNA genome (Havelaar, 1993; Hsu et

al., 1995). Both F+ RNA coliphages and enteroviruses are excreted by humans and

animals. Coliphages are excreted at all times by many humans and warm-blooded

animals, whereas enteric viruses of human health concern are excreted only by humans

during infection which may last for few days to few weeks (Sobsey et al., 1995). For

these reasons, coliphages are valuable models for the study of human and animal

enteric viruses (Gantzer et al., 1998). In addition, coliphages are easily and rapidly

cultivated in labs which makes them good indicators for the presence of enteric viruses

in water, food, shellfish, and wastewater (Furuse, 1981; Sobsey et al., 1995).

To identify conjugative plasmids present in biosolids, Qβ phages can be used. Qβ

phage belongs to the family Leviviridae. The phage genome is surrounded by a cubic or

icosahedral protein capsid without a tail. Qβ has a single-stranded RNA genome of

4,217 bases that encodes four genes for A2, A1, coat protein and the RNA replicase β

subunit (Duin, 1988; Klovins et al., 1998). The host for Qβ is male specific (F+) E.coli cells.

23

Phage infection starts by attachment of Qβ to the E.coli F pilus followed by the release

of phage RNA into the host cell (Figure 5). After the release, phage directs the host

machinery to make copies of the phage RNA and to form a coat protein (Takeshita et al.,

2012; Tsukada et al., 2009). The phage heads are then assembled with the newly

synthesized RNA followed by the release of phages by rupture or lysis of the bacterial

cell within as little as 1 hour after infection (Figure 5). The newly released phages go and

attach to other bacterial cells carrying F-factor and the process continues. These phages

typically produce clear plaques on a lawn of susceptible host bacteria (Davis et al., 1990;

Takeshita et al., 2012; Tsukada et al., 2009). Thus, this technique can be used to

distinguish between cells carrying conjugative and non-conjugative plasmids (Miller,

1972).

24

Figure 5: Infection of RNA phage and cell lysis. RNA phages such as Qβ attaches to the host bacteria through F pili and inserts their genome into the host. The phage RNA then uses the host machinery for making copies of their genome and protein coat. The newly synthesized RNAs are then packed into empty phage heads and the fully packed heads or virions are released by lysing the host cell. The released phages go and attach to other bacterial cells and the process continues.

1.12 Mechanism of Conjugation:

Once again, the plasmids involved in HGT are of two types: (i) self-transmissible or

conjugative, and (ii) nonconjugative or mobilizable plasmids (Salyers and Amabile-

Cuevas, 1997). The self-transmissible plasmids carry necessary genes (tra or transfer) to

25

promote their gene transfer. However, the mobilizable plasmids lack transfer function

(tra genes) but contain an ori site (a specific sequence that allows for their transfer), so

in the presence of conjugative plasmids they are able to transfer their genes to a

recipient (Freifelder, 1987).

The mobility of plasmids can be examined by the conjugation process which requires

a physical contact contact between donor and recipient cells. The donor cell is referred

to as 𝐹+ because it carries a conjugative plasmid for conjugation, whereas the recipient

cell lacking a conjugative plasmid is called the 𝐹−cell (Moat et al., 2002). The F plasmid

encodes a sex pilus, a protein appendage which recognizes and binds to receptors on a

recipient bacterial cell wall. The cell membrane of donor and recipient fuses together

and creates a passage between bacteria for DNA transfer. Then, a plasmid encoded

endonuclease cleaves the plasmid at a specific site called the origin of transfer (ori T).

After that, the 5' end of a single stranded DNA starting from the ori T enters a recipient

𝐹−cell (Phornphisutthimas et al., 2007). Later, complementary strands are synthesized

in both (𝐹+and 𝐹−) bacteria by a rolling circle mechanism. It has been shown when

𝐹+ and 𝐹−cells are mixed together that eventually all the cells will become 𝐹+ (Moat et

al., 2002). The processes involved in conjugation are depicted in Figure 6. Hence,

conjugative plasmids can be identified by conjugative transfer or by their susceptibility

to male-specific coliphages such as Qβ and M13 phages (see section 1.11). If the

plasmids are non-conjugative, they can be identified by a tripartite mating process

which is described below.

26

Tripartite mating is similar to conjugation and can be used to identify non-conjugative

or mobilizable plasmids. As mentioned earlier, non-conjugative plasmids are not able to

make direct gene transfer because they lack transfer function (tra genes) but these

plasmids carry an ori site (a specific sequence that allows for their transfer), so in the

presence of conjugative plasmids they are able to transfer their genes to a recipient cell

(Freifelder, 1987). In tripartite mating, there will be three strains involved: donor, helper

and recipient. The helper strain carry tra genes for transfer function, so it will help the

donor to transfer the plasmid to the recipient cell. It has been shown that in the

presence of bacteria carrying tra genes, non-conjugative plasmids are able to transfer

their genes to recipient bacteria (Droge et al., 2000; Schluter et al., 2007). Thus, non-

conjugative plasmids can be identified by the tripartite mating process.

27

Figure 6: Mechanism of conjugation in bacteria. The donor cell contains an F plasmid which encodes an F pilus for conjugation. The F pilus is a protein appendage which recognizes and binds to receptors on a recipient cell wall, and then the cell wall fuses together and creates a passage between the two bacteria for plasmid DNA transfer.

28

1.13 Objectives:

The goal of this study was to (1) examine the presence of drug resistant bacteria in

Cheney biosolids, (2) to screen them for the presence of resistance (R) plasmids, (3) to

verify the location of resistance genes on R-plasmids, and (4) to identify whether the R-

plasmids are of a conjugative or a non-conjugative type.

29

2. Materials and Methods:

2.1 Bacterial Growth Media:

For growth and isolation of bacteria both liquid and solid media were used. The media used

were: MacConkey agar and Tryptic soy agar (TSA) (Difco, Detriot, MI), Luria-Bertani (LB) agar and

LB broth (Mo-bio, Carlsburg, CA). MacConkey agar was prepared by adding 50g of powder per

liter of deionized water and boiled to dissolve completely. TSA was prepared by dissolving 40 g

of powder in a liter of deionized water. LB broth was prepared by adding 20g of powder per liter

of deionized water. LB agar was prepared by mixing 35 g of powder in a liter of deionized water.

All media were sterilized by autoclaving at 121°C and 15 psi for 15 minutes.

2.2 Preparation of Antibiotic Stock Solutions:

Antibiotics added to the growth media (either broth and agar plates) were made from the

following powdered antibiotics: Ampicillin sodium salt, Tetracycline hydrochloride, Kanamycin,

Nalidixic acid, Streptomycin sulfate salt, Chloramphenicol, Rifampin. All antibiotics were

purchased from Sigma-Aldrich, St. Louis, MO. The antibiotic stock solutions were prepared as

recommended by Sambrook et al., 1989.

All antibiotic solutions were prepared with sterile deionized water in 1.5 ml sterile Eppendorf

tubes. Ampicillin stock solution (100 mg / ml) was prepared by dissolving 100 mg of ampicillin

sodium salt in 1 ml of deionized water in a 1.5 ml Eppendorf tube. Tetracycline stock solution

(15 mg / ml) was prepared by adding 15 mg of Tetracycline hydrochloride to 1 ml of 50%

Ethanol. Kanamycin solution (50 mg / ml) was prepared by dissolving 50 mg of Kanamycin in 1

ml of deionized water. Nalidixic acid solution (30 mg / ml) was prepared by dissolving 30 mg of

Nalidixic acid in 1 ml of 1M NaOH. Streptomycin solution (100 mg / ml) was prepared by mixing

30

100 mg of Streptomycin sulfate salt in 1 ml of deionized water. Chloramphenicol stock solution

(20 mg / ml) was prepared by adding 20 mg of Chloramphenicol to 1 ml of 95% Ethanol.

Rifampin solution (100 mg / ml) was prepared by mixing 100 mg of Rifampin in 1 ml of Methanol

and dissolved by adding 3 drops of 10N NaOH.

All antibiotics were dissolved by vortexing and the mixture was filter sterilized using a 0.2 µm

syringe filter. The filtrate was collected in 1.5 ml sterile Eppendorf tubes and stored at -20°C

until use. From the antibiotic stock solution, appropriate amount was mixed with LB broth and

agar before use. The final concentration of antibiotics used were: ampicillin (100 µg / ml),

tetracycline (15 µg / ml), streptomycin (100 µg / ml), kanamycin (50 µg / ml), rifampin (100 µg /

ml), nalidixic acid (30 µg / ml) and chloramphenicol (20 µg / ml). These final concentrations were

determined based on a recommendation by Sambrook et al. (1989).

2.3 Preparation of Buffer and Solutions:

Dulbecco’s phosphate buffered saline (PBS) was prepared by mixing the following: 13.7 mM

NaCl, 0.27 mM KCl, 1 mM Na2HPO4 and 0.2 mM KH2PO4 in 800 ml of deionized water. The pH

was adjusted to 7.4 with HCl and the final volume was adjusted to 1 liter with additional water.

Saline solution was prepared by dissolving 0.85 g of NaCl in 100 ml of deionized water. The PBS

and Saline were sterilized by autoclaving at 121°C and 15 psi for 15 minutes before use. Both

PBS buffer and saline solution (0.85 %) were used for making serial dilutions of biosolid samples.

The gel electrophoresis buffer, Tris-Acetate EDTA (TAE) was prepared by mixing 0.04 M Tris-

acetate and 0.001 M Ethylenediaminetetraacetic acid (EDTA), disodium salt in 800 ml of

deionized water. The pH was adjusted to 8.0 with acetic acid and the final volume was adjusted

to 1 liter with additional water. The TAE buffer was used for running an agarose gel

electrophoresis.

31

2.4 Collection of Biosolids and Inoculation onto Selective Media:

Biosolid samples were collected from a wastewater treatment facility, Cheney, WA. Roughly

500 g of biosolids were collected in sterile glass bottles. They were placed on ice and

transported to lab and stored in a 4°C cold room until use. From that, 10 g of biosolids were

suspended in 90 ml of PBS and placed on a rotary shaker for 3 hours. The liquid was collected

and serial dilutions (10-2 to 10-7) were made. The diluted sample (100 µl) was spread on

MacConkey agar and incubated at 37°C for 24-36 hours. Single isolated colonies were picked

randomly with sterile toothpicks and streaked on antibiotic plates, and the plates were

incubated at 37°C for 36-48 hours. Resistant bacterial strains were once again tested on the

antibiotic plates using the same procedure to ensure that the isolates were truly resistant to the

antibiotics used.

2.5 Bacterial Strains Used:

The bacterial strains and phages used in this study were: Escherichia coli MM294, E. coli

DH5α, E. coli XK1502, E. coli MC4100 and phages Qβ and M13. The source and genotype for

these organisms are shown in Table 4a and 4b.

32

Table 4a:

Control

Bacteria

Strain

Designation Genotype Source

Escherichia coli MM294 F -, glnV44(AS), λ-, rfbC1, endA1, spoT1,

thiE1, hsdR17, creC510

Coli Genetic Stock Center

(CGSC) #:6315

Escherichia coli DH5α F -, φ80lacZΔM15 Δ(lacZYA-argF)U169,

recA1 endA1, hsdR17(rk-, mk+),

phoAsupE44 thi-1, gyrA96, relA1 λ-

Biology department

collection

Escherichia coli MM294

transformed

with pGLO

F -, glnV44(AS), λ-, rfbC1, endA1, spoT1,

thiE1, hsdR17, creC510

Biology department

collection

Escherichia coli MC4100 F -, [araD139]B/r, Δ(argF-lac)169, λ-, e14-,

flhD5301, Δ(fruK-yeiR)725(fruA25), relA1,

rpsL150(strR), rbsR22, Δ(fimB-

fimE)632(::IS1), deoC1

Dr. Steve Moseley,

Microbiology

Department, UW

Escherichia coli XK1502 F', lacU169, traD8, nalA Dr. Steve Moseley,

Microbiology

Department, UW

Table 4b:

Control Phage Strain Designation Source

RNA Phage

Qβ

Dr. Steve Moseley, Microbiology

Department, UW

Single-stranded (ss) DNA phage M13 From our collection

33

2.6 Long Term Storage of Bacterial Isolates:

Resistant bacterial isolates were stored at -80°C in LB broth containing 15% (v/v) glycerol

(Sigma Aldrich, St. Louis, MO). Bacterial isolates were streaked on LB plates containing

antibiotics and incubated at 37°C for 24 hours to get individual colonies. A single isolated colony

was picked and transferred to a 5ml LB broth containing antibiotics and placed on a 37°C shaker

for 18 hours. Then, 0.7 ml of cell suspension was transferred to sterile 1.2 ml Cryovial (Midsci™,

St. Louis, MO) and mixed with 0.3 ml of sterile 15% (v/v) glycerol. Samples were stored at -20°C

for 48 hours and then moved to -80°C for long-term storage.

2.7 Plasmid DNA Extraction:

Plasmid DNA was extracted from resistant bacteria using the following kits: GeneJETTM

Plasmid Miniprep Kit (Fermentas Life Sciences, Burlington, Ontario) and ZyppyTM Plasmid

Miniprep Kit (Zymo Research, Irvine, CA). Plasmid DNA was extracted from resistant bacteria

and their size and concentration were determined using 0.7% (w/v) agarose gel electrophoresis.

As per the manufacturer’s protocol, an isolated colony was picked from an antibiotic plate

and transferred to a 5 ml LB broth containing antibiotics and placed in a 37°C shaker for 16-18

hours. Then, 3 ml of cell suspension was transferred to two 1.5 ml sterile Eppendorf tubes and

centrifuged at 10,000 X G for 4 minutes at room temperature. The supernatant was discarded

and the cells were suspended with SDS/alkaline lysis buffer (supplied in kit) to release plasmid

and genomic DNA. The resulting lysate was neutralized using neutralization buffer from the kit.

Cell debris, SDS precipitate and the genomic DNA trapped in them were pelleted by

34

centrifugation and the supernatant containing the plasmid DNA was loaded onto the spin

column membrane for further purification. The adsorbed DNA was washed to remove

contaminants using wash buffer supplied in the kit. Finally, the plasmid DNA was eluted with

elution buffer (from kit) and the DNA was stored at -20°C until use.

2.8 Agarose Gel Electrophoresis:

The agarose gel electrophoresis was used for the separation and visualization of plasmid DNA

and also to determine the size and concentration of the DNA. The materials used for that

purpose were: agarose (Invitrogen, Eugene, OR), TAE buffer, an electrophoresis chamber (Owl

model B1A class II, Owl Separation Systems, Portsmouth, NH), 6X loading dye (New England

Biolabs, Ipswich, MA) containing 5X SYBR gold (Invitrogen, Carlsbad, CA), Supercoiled DNA

ladder (2-10 kb)(Cat#N04725, New England Biolabs, Ipswich, MA), 1 kb DNA ladder (0.5-

10kb)(Cat#N3232S, New England Biolabs, Ipswich, MA), an Ultraviolet Transilluminator

(Ultraviolet Products, Upland, CA) and Kodak EDAS 290 (Kodak, Rochester, NY) imaging system.

A 0.7% (w/v) gel was prepared by dissolving 0.35 g of agarose (Invitrogen, Eugene, OR) in 50

ml of TAE buffer and poured into an electrophoresis chamber (Owl model B1A class II, Owl

Separation Systems, Portsmouth, NH). Samples (3 μl) were mixed with 1 μl of 6X loading dye

(New England Biolabs, Ipswich, MA) containing 5X SYBR gold (Invitrogen, Carlsbad, CA) and the

total mixture (4 μl) loaded into wells and allowed to run at 58-volt for 3 hours. A (2-10 kb)

Supercoiled DNA ladder (Cat#N04725 New England Biolabs, Ipswich, MA) was used to determine

the size and concentration of plasmid DNA. The DNA within the gel matrix was visualized with an

Ultraviolet Transilluminator (Ultraviolet Products, Upland, CA) and photographed with a Kodak

EDAS 290 (Kodak, Rochester, NY) imaging system. The size and concentration of the sample DNA

were measured by comparing visually the fluoresecence of sample DNA band(s) with the

35

standard DNA ladder. Based on the size of sample DNA band(s) their corresponding

concentrations were determined using the standard DNA concentration chart provided by the

supplier.

2.9 Identification of Bacteria:

The bacterial isolates were identified using API-20E system and oxidase test. The materials

used were: 0.85% saline solution, TSA plates, mineral oil, sterile tooth picks, sterile filter papers

(Whatman No.1), API strips (BioMerieux, Durham, NC) and Oxidase reagent (Becton, Dickson

and Company, Sparks, MD). Bacteria were grown on TSA plates at 37°C for 18-24 hours. A single

well-isolated colony was picked and suspended in 0.85% saline solution and mixed thoroughly.

Using a sterile pipette, bacterial suspension was added into the cupules of API strips and then

mineral oil was added in recommended cupules and the strips were incubated at 37°C for 24-36

hours. Later, other reagents (BioMerieux supplies) were added and the color change was

recorded as (+) and (–) signs. Based on the (+/-) score, an organism was identified using the

Analytical Profile Index (BioMerieux, Durham, NC, 1999). For oxidase test, few drops of Oxidase

test reagent (Becton, Dickson and Company, Sparks, MD) were added to a strip of Whatman

filter paper. A single isolated colony was streaked on top of the reagent using a sterile toothpick

and the color change from white to dark blue is considered as positive and no color change was

considered as negative for the test.

2.10 Preparation of Competent Cells:

The competent cells were prepared as described in the MicroPulser lab manual (Bio-Rad,

Hercules, CA). The E. coli MM294 was grown in a 5 ml LB broth on a 37°C rotary shaker for 18-24

hours. Then, 500 ml of fresh LB broth was inoculated with 5 ml of overnight E. coli culture and

36

grown on a 37°C shaker till the culture had reached the cell density of 0.3-0.5 at A550 using the

SHIMADZU UV-1201 spectrophotometer (Columbia, MD). The culture was chilled on ice for 30

minutes and transferred to ice-cold centrifuge tubes and spun at 4000 X G for 15 minutes at 4°C

using a Sorvall RC5B refrigerated centrifuge (Sorvall, Newtown, CT). The supernatant was

discarded and the bacterial pellet was suspended with 500 ml of ice-cold MilliQ® water and

spun at 4000 X G for 15 minutes at 4°C. This step was repeated twice with 250 ml ice-cold

MilliQ® water and centrifuged in the same condition. The bacterial pellet was suspended in 20

ml ice-cold 10% (v/v) glycerol and spun at the same condition. Finally, the cell pellet was

suspended in 2 ml of ice-cold 10% (v/v) glycerol and divided into small aliquots (50 µl) in 0.5 ml

sterile Eppendorf tubes and stored at -80°C until needed.

2.11 Transformation:

Electroporation was carried out using a MicroPulser (Bio-Rad, Hercules, CA). The following

items were used for that purpose: Super Optimal Broth (SOB) containing 10mM glucose,

electroporation cuvette (0.2 cm gap), LB plates with appropriate antibiotics and a 37°C shaker.

The electro-competent cells were retrieved from a freezer and thawed on ice. Cell suspension

(50 µl) was mixed with either 0.2 µg of pBR322 (positive control) or 0.3 µg of plasmid DNA

extracted from the isolates. The mixture of cells and DNA were incubated on ice for 2 minutes

and transferred to a cold electroporation cuvette. This mixture was subjected to one 2.5 kV

pulse for 5 milli sec using a Bio-Rad MicroPulser (provided by Dr. Andrea Castillo, EWU Biology

Dept). After a pulse, 1 ml of SOB medium was added to the cuvette and the entire content was

transferred to a 5 ml test tube and incubated on a 37°C rotary shaker for 2 hours. Then, 100 µl

of the cell suspension was spread on antibiotic plates and incubated at 37°C for 36-48 hours.

The bacterial colonies or transformants formed on these antibiotic plates were tested again on

37

the same antibiotic to confirm their drug resistance. Later, plasmids were extracted from the

transformants using the same procedure as mentioned in the section ‘plasmid DNA extraction’.

The presence of plasmids and their size and concentration were determined on an agarose gel

following the same procedure. Plasmids extracted from the transformants were compared with

the original plasmids used for transformation on an agarose gel to verify similarities between

these two plasmids.

2.12 Restriction Digestion:

Restriction digestion was performed with the plasmids to confirm their size(s) after linearizing

them. Plasmid DNA extracted from the transformants was digested with the restriction enzyme

EcoRI (#FD0274, Fermentas, Glen Burnie, MD). The materials used were: sterile MilliQ® water,

10X FastDigest buffer (10mM potassium phosphate, 300mM Nacl, 1mM EDTA, 1mM DTT,

0.2mg/ml BSA, 0.15% Triton X-100 and 50% (v/v) glycerol), sterile 0.5 ml sterile Eppendorf tubes,

1 kb DNA ladder (N3232S, New England Biolabs, Ipswich, MA) and a 37°C heat block.

The reaction mixture (20 µl) was prepared by mixing 15 µl of sterile MilliQ® water, 2 µl of

10X FastDigest buffer, 1 µg of plasmid DNA and 10 units of EcoRI FastDigest enzyme in 0.5 ml

sterile Eppendorf tubes. The mixture was spun at 3000 X G for a minute and incubated in a 37°C

heat block for 3 hours. The digested DNA or 1kb DNA ladder were then mixed with 3 µl of 6X

SYBR gold containing dye and loaded on a 0.7% (w/v) agarose gel and electrophorized.

2.13 Assay of Phage Sensitivity:

Phage sensitivity assay was performed to determine the presence of conjugative plasmid in

the isolates. The materials used were Qβ and M13 phages, Ecoli MC4100 and XK1502, 15 ml

38

centrifuge tubes, LB broth, LB plates and chloroform (Sigma Aldrich, St. Louis, MO). The

procedure for phage sensitivity was followed as suggested by Miller (1972).

The Qβ and M13 phage lysate was prepared as follows: E. coli XK1502 were grown in a 5 ml

LB broth on a 37 °C rotary shaker for 18-24 hours. The overnight culture was diluted (1:100)

with 5ml of fresh LB broth and allowed to grow for 3 hours on a 37°C rotary shaker. The Qβ and

M13 phages were added to the bacterial cell suspension in two separate tubes in a ratio 1:10

and incubated in a 37°C water bath overnight. Three drops of chloroform were added to the cell

suspension and centrifuged at 5000 X G for 10 minutes and the supernatant was carefully

removed and stored at 4°C until use. The clear supernatant containing phages (phage lysate)

was further used in phage sensitivity assay. The test isolates or E.coli XK1502 (positive control)

or E.coli MC4100 (negative control) were grown to exponential phase and 100µl was plated on

LB agar and let it dry for an hour. Then, 5µl of phage lysate was spotted on the LB plate

containing test bacteria or positive control E.coli XK1502 or negative control E.coli MC4100 and

the plates were incubated at 37 °C for 24 hours. The visible plaques formed on plates indicate

the presence of conjugative plasmid in bacteria.

2.14 Tripartite Mating:

Tripartite mating process was used to identify non-conjugative but mobilizable plasmids. The

materials used were: LB broth, LB plates containing antibiotics, control E.coli strains MC4100

and XK1502, a 37°C shaker. The procedure for tripartite mating was followed as per Miller

(1972).

The tripartite mating process involve three different strains: a donor, a helper and a recipient.

The donor is the strain isolated from biosolids, helper is the strain of E.coli XK1502 and the

39

recipient is the E.coli MC4100 strain. All the three strains were streaked on suitable antibiotic

plates to get isolated colonies and incubated at 37°C for 24 hours. A single well-isolated colony

from each plate was picked and transferred to a 5 ml LB broth and incubated at 37°C for 18-24

hours. The overnight culture was diluted with 5 ml of fresh LB broth in the ratio 1:40 for donor

and helper, and 1:20 for recipient and allowed to grow for 3 hours on a 37°C rotary shaker. All

the antibiotic plates were divided into four sections: 1. Donor (biosolid isolate), 2. Helper (E.coli

XK1502), 3. Recipient (E.coli MC4100), and 4. Donor + Helper + Recipient. The recipient culture

(5 µl) was first spotted on appropriate sections and let it dry for 30 minutes. The donor (5 µl)

was then spotted directly on top of the recipient and let them dry for another 30 minutes.

Finally, the helper (5 µl) was spotted on top of the recipient and donor spots and allowed to

completely dry for 30 more minutes, and the plates were incubated at 37°C for 24 hours. The

transconjugants or bacterial colonies formed on the antibiotic plates in a sector in which all

three types were mixed together were then evaluated. Conjugation (bi-partite mating) was also

performed simultanoeusly at the same time of tripartite mating to check whether the biosolid

isolate carried conjugative plasmid. Conjugation is similar to tripartite mating except there are

only two strains involved. The antibiotic plates were divided into three sections: 1. Donor

(biosolid isolate), 2. Recipient (E.coli MC4100) and 3. Donor + Recipient. The diluted fresh

cultures of donor and recipient as used in tripartite mating were used in this assay. The recipient

culture (5 µl) was first spotted on appropriate sections and let it dry for 30 minutes. The donor

(5 µl) was then spotted directly on top of the recipient and let it dry for another 30 minutes and

the plates were incubated at 37°C for 24 hours. The transconjugants or bacterial colonies

formed on the antibiotic plates were then evaluated.

40

3. Results and Discussion:

3.1 Collection of Biosolids and Isolation of Resistant Bacteria:

Wastewater serves as a reservoir of bacteria and viruses that may contribute to the

selection and transfer of antibiotic resistance genes to other bacteria (Binh et al., 2008;

Lund et al., 1996). Biosolids are the final product of the waste water treatment process,

and they are used as fertilizer to promote plant growth all over the United States. They

are used in public parks, agricultural lands and home gardens (U.S. EPA, 1994). People

can be directly or indirectly affected by such application. For example, bioaerosols or

microbes can be dispersed through wind, and people can inhale these organisms and

get infected (Baerisch et al., 2007). Pathogens can enter the human food chain by

consuming food crops and vegetables grown on biosolid applied lands (Tamminga et al.,

1978; Wei et al., 2010). Farm animals grazing on biosolid applied lands also contribute

to the spread of pathogens to humans who consume meat products. It has been shown

that addition of organic wastes to soil in the form of biosolids increases the growth and

survival efficiency of E.coli and others (Unc et al., 2006). Moreover, several studies

reported the presence of drug resistant bacteria and viruses in biosolid samples (Binh et

al., 2008; Wong et al., 2010). To my knowledge, Cheney biosolids are tested primarily

41

for the reduction of Salmonella and fecal coliforms such as E.coli, Citrobacter,

Enterobacter and Klebsiella. It is not clear whether these biosolids are tested for other

pathogens like drug resistant bacteria and viruses. In this study, we investigated the

presence of drug resistant bacteria and their role in the transfer of drug resistance

genes to other bacteria.

Biosolids were collected in June, 2012 from the wastewater facility, Cheney, WA. The

samples were diluted and plated on MacConkey agar to select for gram-negative

bacteria. From these plates, 100 colonies of varied colony morohology were randomly

picked and tested against seven antibiotics representing a variety of antibiotic classes.

Drugs from six different classes that are commonly used to prevent human infections

were selected for this study. In aminoglycoside class, kanamycin and streptomycin were

used. In the penicillin class, ampicillin was used. Nalidixic acid was selected from the

quinolone class. In the tetracycline class of antibiotics, tetracycline was used. Other

antibiotics such as rifampin and chloramphenicol were also tested. Of the 100 colonies

tested, 68% of bacteria were resistant to two or more drugs (Table 5a and 5b). Of the 68

resistant isolates, 81% were resistant to ampicillin, 35% to kanamycin, 56% to

streptomycin, 35% to chloramphenicol, 62% to nalidixic acid, 51% to tetracycline and

68% to rifampin (Figure 7). Overall, many of the bacteria screened were ampicillin and

rifampin resistant which suggests the high usage of these antibiotics in the community

(Figure 7). Ampicillin resistant isolates frequently exhibited tetracycline, streptomycin

and rifampin resistance which is consistent with the study of others (Mirzaagha et al.,

2009; Van Donkersgoed et al., 2003). We also found chloramphenicol resistant isolates

42

which is surprising because chloramphenicol is withdrawn in U.S based on their toxic

effects. Macrolide antibiotics such as erythromycin inhibits protein synthesis by binding

to 50S ribosomal subunit similar to chloramphenicol. Erythromycin resistant isolates

have been isolated and identified from Cheney waste water facility (Marshall, 2009).

This could be the reason for the presence of chloramphenicol resistance among

bacteria. In addition, studies show resistance (R) plasmid that confer ampicillin and

tetracycline resistance frequently confer chloramphenicol resistance (Haldar et al.,

1995; Mandal et al., 2005). We found many ampicillin and tetracycline resistant isolates

which also carried plasmid, so this might have conferred chloramphenicol resistance in

bacteria. Of the 68 resistant isolates, 17.6% were resistant to two drugs, 20.5% to three

drugs, 26.4% to four and five drugs and finally 8.8% were resistant to six drugs. None of

the isolates were resistant to all the drugs tested (Figure 8). In this study, we found

bacteria that were resistant to penicillin, tetracycline and quinolone class of antibiotics

which is consistent with the previous reports (Marshall, 2009).

43

Table 5a: Resistance profile of the isolates X1-X36

Isolate # Amp Kan Str Chl Nal Tet Rif X1 + - + - + + - X2 + + + - - + + X3 + - - + - - + X4 + + + - + + - X5 + + + + + + - X6 + - - - - - + X7 + - - + - + - X8 + - + + + - + X9 + - + - - - +

X10 + - - + - - + X11 + - + - + + - X12 + + - + + - - X13 + - - + - - + X14 + + - + - - + X15 + - - - + - + X16 + - + - - + - X17 + - - - - + + X18 + - - - + - + X19 + - - - + - - X20 + - + + + - + X21 - - + - - - + X22 + - + - - + + X23 + - + + - + + X24 + - + - - + + X25 + + + - - + + X26 + - - + - - - X27 + - + - - + + X28 + - + + - + + X29 + - - - - - + X30 + - + + + + - X31 + + + - - + +

44

X32 + + - - - + + X33 + + + + - + + X34 + + + - + + + X35 + - + - + + - X36 + - + - - + +

Table 5b: Resistance profile of the isolates X37-X68

Isolate # Amp Kan Str Chl Nal Tet Rif X37 + + - - + + + X38 - - + - + - - X39 + - - - + + + X40 + + - - + - + X41 + - + - + + + X42 + + - + + - + X43 + + - - + - + X44 + + + + + - + X45 + + + + + - + X46 + + - - + - - X47 + + + - + - - X48 + + + + - + X49 + + + - + - + X50 + - + - + + + X51 - - - - + - + X52 - - + + + + + X53 + - + - - - + X54 + + + + + - + X55 - - - + + - - X56 + - + - + + + X57 - + + + + - - X58 - - + - - - + X59 - - - - + + - X60 - + + + + + - X61 - - - - + + + X62 + - - - + + + X63 - - - - + + - X64 + - + - + + - X65 - - - + + + - X66 + - - - + + + X67 + - - - + - +

45

X68 - + - - + - -

Figure 7: The graph represents the percent of isolates that were resistant to different antibiotics.

0

10

20

30

40

50

60

70

80

90

Amp Kan Str Chl Nal Tet Rif

Perc

ent (

%) R

esis

tanc

e

Antibiotics Used

46

Figure 8: The graph represents the percent of isolates that were resistant to a given number of the antibiotics tested.

We did not do any correlation study regarding the usage of antibiotics by the local

community and the presence of drug resistant bacteria in Cheney biosolids. Former

student in our lab took survey from three pharmacies in Cheney local, and tried to do

some correlation study (Marshall, 2009). However, she was not able to correlate the

antibiotics sold by local pharmacies with the drug resistant bacteria present in Cheney

biosolids (Marshall, 2009). Many people in Cheney commute to Spokane every day and

vice versa, so they could obtain antibiotics from pharmacies outside the city limit. It is

difficult to survey the amount of prescription antibiotics sold by all these pharmacies

because of privacy issues. Amount of resistant bacteria and unadsorbed antibiotics

0

5

10

15

20

25

30

2 3 4 5 6 7

Perc

ent (

%) I

sola

tes

Resistance to Number of Drugs

47

excreted by the people can also vary (Sarmah et al., 2006). For these reasons, antibiotics

that are commonly used by people are chosen for this study.

3.2 Isolation of Plasmids from Resistant Bacteria:

Multi-drug resistant bacteria are more likely to carry resistance plasmids (Nikaido,

2009; Maruyama et al., 2006). To verify this possibility, plasmids were extracted from all

the 68 resistant isolates X1 to X68 (data not shown here). Prior to plasmid extraction,

resistant isolates were once again streaked on antibiotic plates to ensure their drug

resistance (Figure 9). Using the manufacturer’s protocol of plasmid isolation kit,

plasmids were isolated, and their size and yield were determined on a 0.7% agarose gel.

There were nine isolates X3, X9, X11, X16, X41, X52, X53, X58 and X66 that showed the

presence of plasmids (Figure 10a, 10b and Table 6). Here, we showed that multi-drug

48

resistant bacteria carried resistance plasmid which is consistent with other studies

(Bergstrom et al., 2000; Kruse and Sorum, 1994; Nikaido 2009). There are two possible

ways to explain the absence of plasmids in the remaining resistant isolates. One, the

resistant bacteria carry resistance genes on their chromosome. It has been shown that

drug resistance genes are often encoded on the bacterial chromosome (Carattoli, 2001;

Drlica and Malik, 2003; Sharma and Mohan, 2006). Second, the resistant bacteria might

have carried large molecular weight plasmids which were not processed in this assay.

The plasmid preparation kit used was suitable for smaller size plasmids (<20 kb), so

plasmids larger than that might not be isolated (Fermentas Life Sciences, Irvine, CA).

49

Figure 9: Antibiotic sensitivity testing of the isolates. At the top from left to right: the plates contain ampicillin, rifampin, kanamycin and tetracycline. At the bottom from left to right: the plates contain chloramphenicol, streptomycin and nalidixic acid. These antibiotic plates serve as a model to represent the sensitivity of bacteria to various drugs.

50

Figure 10a: Agarose gel showing the presence of plasmids.

Lane C-, E.coli DH5α lacking a plasmid. Lane L is a supercoiled DNA ladder. Lane C+, E.coli MM294 containing pGLO plasmid. Lanes X3, X9, X11, X16 and X41 are the isolates that showed the presence of plasmids.

Figure 10b: Agarose gel showing the presence of plasmids.

Lane C-, E.coli DH5α lacking a plasmid. Lane L is a supercoiled DNA ladder. Lane C+, E.coli MM294 containing pGLO plasmid. Lanes X52, X53, X58 and X66 are the isolates that showed the presence of plasmids.

51

3.3 Identification of Resistant Bacteria:

The nine isolates (X3-I, X9-I, X11-I, X16-I, X41-I, X52-I, X53-I, X58-I and X66-I) that

showed the presence of plasmids were identified using the API identification scheme

(Figure 11) and oxidase test. The three isolates (X3-I, X52-I and X58-I) were identified as

Escherichia coli, two isolates (X9-I and X66-I) as Salmonella spp and two isolates (X16-I

and X53-I) as Kluyvera spp. There was one isolate (X11-I) identified as Enterobacter

aerogenes. Finally, the remaining isolate (X41-I) could be either Klebsiella pnuemoniae

or Klebsiella planticola. These data are summarized in Table 6. The isolates were

identified based on some important biochemical tests. For instance, E. coli isolates were

positive for indole production, beta-galactosidase and ornithinine decarboxylase

reactions. The Klebsiella pnuemoniae isolates were positive for citrate utilization, urea

hydrolysis and acetoin production. Similarly, Kluyvera isolates were positive for indole

production, citrate utilization and glucose oxidation; the Salmonella isolates were

positive for ornithinine decarboxylase and hydrogen sulfide production. Finally, the

Enterobacter aerogenes was positive for citrate utilization, ornithinine decarboxylase

and acetoin production. None of the isolates were positive for the oxidase test

confirming that these isolates truly belong to Enterobacteriaceae family.

52

Figure 11: API-20E strips used for the identification of bacteria. At the top is the isolate X58-I identified as E.coli. At the middle is an isolate X16-I identified as Kluyvera spp. At the bottom: the isolate X11-I identified as Enterobacter aerogenes.

Table 6: Identity of resistant bacteria and their antibiotic resistance profile

Isolate # Antibiotic Resistance API Identification

X3-I Amp, Chl, Rif E.coli

X9-I Amp, Str, Rif Salmonella spp.

X11-I Amp, Str, Nal, Tet Enterobacter aerogenes

X16-I Amp, Str, Tet Kluyvera spp.

X41-I Amp, Str, Nal, Tet, Rif K.pnueumoniae

/K.planticola

X52-I Str, Nal, Tet, Rif, Chl E.coli

X53-I Amp, Str, Rif Kluyvera spp.

X58-I Str, Rif E.coli

X66-I Amp, Tet, Nal, Rif Salmonella spp.

53

The API and oxidase test results indicated that all the bacteria identified (E.coli,

Salmonella, Enterobacter, Kluyvera and Klebsiella) belong to Enterobacteriaceae family

which is consistent with the results of others (Hoyle et al., 2005; Van Donkersgoed et al.,

2003). Studies show that members of the Enterobacteriaceae family are frequently

found in sewage samples because many of them are present in the human and animal

gastro-intestinal tract (Hoyle et al., 2005; Van Donkersgoed et al., 2003). A correlation

between antimicrobials used and the development of drug resistance in E.coli has been

well documented (Van den Bogaard and Stobberingh, 2000). It has been shown that

resistance determinants can be transferred from E.coli to enteric pathogens (Aslam and

Service, 2006; Blake et al., 2003). In this study, we identified multidrug resistant E.coli

and other organisms. Many of these multidrug resistant organisms carried plasmid

which is in agreement with the previous reports (Bergstrom et al., 2000; Nikaido 2009).

Furthermore, it has been reported that conjugative plasmids encoding multidrug

resistance genes could be responsible for the transfer of resistance among

Enterobacteriaceae (Paterson, 2006 and Poppe et al., 2001).

54

3.4 Transformation:

Transformation was performed to identify the location of resistance genes. Plasmids

isolated from nine isolates (X3-I, X9-I, X11-I, X16-I, X41-I, X52-I, X53-I, X58-I and X66-I)

were introduced into E.coli MM294 (Figure 10a and 10b). These 9 plasmids are referred

to as ‘parental plasmids’ in this work. The bacterial colonies formed on the antibiotic

plates represent transformants that took the plasmid DNA. Of the 9 plasmid isolates,

only 7 plasmids (77.7%) were successfully and experimentally introduced into E.coli

MM294 (Table 7). The transformation efficiency of these seven isolates was in the range

of 6.7 X 102 to 4.7 X 103 colony forming units per microgram of DNA (shown in Table 7).

Based on transformation results, it is clear that tetracycline, ampicillin, streptomycin,

nalidixic acid and rifampin resistance is carried on a plasmid (Table 7) which is consistent

with other studies (Roberts, 2006). The resistance of these transformants was

reconfirmed by streaking them on the appropriate antibiotic plates. Plasmids were then

reisolated from 2-3 randomly selected transformants, and their presence was confirmed

on an agarose gel (Figure 12a – 12f).

55

Table 7: Transformation efficiency of Parental Plasmids

Transformant Isolate #

Growth of Transformants on Antibiotic Plates No of Transformants / μg DNA

X3-II No growth -

X9-II Amp 3.4 X 𝟏𝟎𝟑

X11-II Str, Tet

Str – 1.7 X 𝟏𝟎𝟑 Tet – 3.4 X 𝟏𝟎𝟑

X16-II Tet 3.2 X 𝟏𝟎𝟑

X41-II Rif 7.4 X 𝟏𝟎𝟐

X52-II Nal, Str, Tet

Nal - 6.7 X 𝟏𝟎𝟐 Str – 3.7 X 𝟏𝟎𝟑 Tet – 4.7 X 𝟏𝟎𝟑

X53-II Rif 6.7 X 𝟏𝟎𝟐

X58-II No growth -

X66-II Tet 3.0 X 𝟏𝟎𝟑

56

Figure 12a: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X9-I plasmid. Lanes 3, 4 and 5 are E.coli transformants taken from an Ampicillin plate. Lane L is a supercoiled ladder. Lanes 7 is a parental X53-I plasmid. Lanes 8 and 9 are E.coli transformants taken from from a Rifampin plate. Lane C+ is E.coli MM294 transformed with pGLO plasmid.

Figure 12b: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X11-I plasmid. Lanes 3, 4, 5 and 7 are E.coli transformants taken from a Streptomycin plate. Lane L is a supercoiled ladder. Lanes 8 and 9 are E.coli transformants taken from a Tetracycline plate. Lane C+ is E.coli MM294 transformed with pGLO plasmid.

57

Figure 12c: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X41-I plasmid. Lanes 3, 4 and 5 are E.coli transformants taken from a Rifampin plate. Lane L is a supercoiled ladder. Lanes 7 is a parental X52-I plasmid. Lane 8 is E.coli transformants taken from a Nalidixic acid plate. Lane C+ E.coli MM294 transformed with pGLO plasmid.

Figure 12d: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X52-I plasmid. Lanes 3 and 5 are E.coli transformants taken from a Tetracycline plate. Lane L is a supercoiled ladder. Lanes 6, 7 and 8 are E.coli

58

transformants taken from a Streptomycin plate.

Figure 12e: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X16-I plasmid. Lanes 3 and 4 are E.coli transformants taken from a Tetracycline plate. Lane L is a supercoiled ladder. Lane C+ is E.coli MM294 transformed with pGLO plasmid.

Figure 12f: A comparison between plasmids isolated from transformants with parental plasmids.

Lane C- is E.coli MM294 lacking a plasmid. Lane 2 is a parental X66-I plasmid. Lanes 3 and 4 are E.coli transformants taken from a

59

Tetracycline plate. Lane L is a supercoiled ladder. Lane C+ is E.coli MM294 transformed with pGLO plasmid.

The transformants (X9-II, X11-II, X16-II, X41-II, X52-II, X53-II and X66-II) showed

resistance patterns similar to the parental strains (Table 6 and 7). For instance, the

parent (X11-I) was resistant to four drugs: ampicillin, streptomycin, nalidixic acid,

tetracycline whereas the transformant (X11-II) was resistant to streptomycin and

tetracycline. That means, the streptomycin and tetracycline resistance were carried on a

plasmid. These two resistance genes could be carried on a same plasmid or two

different plasmids (Table 6 and 7). Similarly, X41-I was resistant to five drugs: ampicillin,

streptomycin, nalidixic acid, tetracycline and rifampin. However, the transformant X41-II

carried rifampin resistance on a plasmid (Table 6 and 7). The remaining or missing

resistance might be carried on a chromosome. Many of the plasmids extracted from

transformants (X41-IIr, X52-II and X16-IIt) migrated at the same distance as parental

plasmids on a gel (Figure 12c, 12d and 12e). There were few exceptions; plasmids X9-IIa,

X11-IIs and X66-IIt extracted from transformants migrated faster than the parental

plasmids (Figure 12a, 12b and 12f). This is not surprising because E. coli will take only

limited amount of DNA during transformation, and it is unlikely that E. coli take more

than one plasmid. The different migration patterns of parent and transformants on the

gel could be because E. coli took only one plasmid eventhough the parent contained

multiple plasmids. Some of the transformants showed the presence of multiple plasmids

with different sizes on a gel. The multiple plasmid bands may reflect the presence of

plasmid in isomeric forms such as supercoiled, relaxed and nicked (Schmidt et al., 1999).

60

To check whether these are truly different forms of a plasmid, we performed restriction

digestion which is described in the next section (Figure 13a – 13g). The plasmids from

two isolates (X3-I and X58-I) failed to transform in the E.coli strain. There could be a

reason for that. The resistance genes might not be present on plasmids which prevented

E.coli from growing on antibiotic plates. These two isolates might have carried

resistance genes on the bacterial chromosome.

3.5 Restriction Digestion:

Restriction digestion was performed to verify whether the multiple DNA bands

represent a single plasmid in different forms. It has been shown that plasmids exist in

various forms and the supercoiled plasmid moves much faster than the linear and

nicked plasmids on an agarose gel (Schmidt et al., 1999). As mentioned in the previous

section, seven out of nine plasmid preparations successfully produced transformants.

These seven isolates were cross checked on antibiotic plates, and plasmids were

extracted from them. These plasmids were further digested with the Type II restriction

enzyme, EcoRI. Many of the isolates X9-II, X11-II, X16-II, X41-II, X52-II, X53-II, X66-II

formed single band on an agarose gel after digestion with EcoRI, suggesting these

plasmids contained a single EcoRI restriction site (Figure 13a, 13c, 13d, 13e, 13f and

13g). Only exception was X11-II, it formed 3 to 4 bands of smaller size than the original

plasmid (Figure 13b). This is not surprising because an EcoRI site is predicted to occur

once every ~4000 bp, and the size of parental plasmid was estimated to be >10 Kb, so it

might have resulted in multiple small fragments.

61

62

Figure 13a: Restriction digestion of the plasmids extracted from transformants.

Lane 1 is an undigested plasmid, pBR322. Lane 2 is a plasmid pBR322 digested with EcoRI shows a single band as expected. Lanes 1 and 2 serve as a positive control. Lane L is a 1 Kb DNA ladder. Lanes 3, 5 and 7 are X9-II plasmid samples that are not subjected to any digestion. Lanes 4, 6 and 8 are the respective plasmids from X9-II that are digested with EcoRI.

63

Figure 13b: Restriction digestion of the plasmids extracted from transformants.

Lane L is a 1 Kb DNA ladder. Lanes 1, 3, 5 and 7 are X11-II plasmid samples that are not subjected to any digestion. Lanes 2, 4, 6 and 8 are the respective plasmids from X11-II that are digested with EcoRI.

Figure 13c: Restriction digestion of the plasmids extracted from transformants.

Lanes 1 and 3 are X11-II plasmid samples that are not digested. Lanes 2 and 4 are X11-II plasmid samples that are digested with EcoRI. Lane L is a 1 Kb DNA ladder. Lanes 5 and 7 are the respective plasmids from X16-II plasmid samples that are not subjected to any digestion. Lanes 6

and 8 are the respective plasmids from X16-II that are digested with EcoRI.

64

Figure 13d: Restriction digestion of the plasmids extracted from transformants.

Lanes 1, 3 and 5 are X41-II plasmid samples that are not digested. Lanes 2, 4 and 6 are X41-II plasmid samples that are digested with EcoRI. Lane L is a 1 Kb DNA ladder. Lane 7 is X52-II plasmid sample that is not subjected to any digestion. Lane 8 is the respective plasmid from X52-II digested with EcoRI.

Figure 13e: Restriction digestion of the plasmids extracted from transformants.

Lanes 1, 3, 5 and 7 are X52-II plasmid samples that are not digested. Lanes 2, 4, 6 and 8 are the respective plasmids from X52-II that are digested with EcoRI. Lane L is a 1 Kb DNA ladder.

65

Figure 13f: Restriction digestion of the plasmids extracted from transformants.

Lane 1 is an undigested plasmid, pBR322. Lane 2 is a plasmid pBR322 digested with EcoRI. Lanes 1 and 2 serve as a positive control. Lane L is a 1 Kb DNA ladder. Lane 3 is an X66-II plasmid sample digested with EcoRI. Lane 4 is an X66-II plasmid sample that is not digested. Lane 5 is X66-II plasmid sample that is not digested. Lane 6 is the respective plasmid from X66-II digested with EcoRI.

Figure 13g: Restriction digestion of the plasmids extracted from transformants.

Lane 1 is an X52-II plasmid sample that is not digested. Lane 2 is an X52-II plasmid sample digested with EcoRI. Lane L is a 1 Kb DNA ladder. Lanes 3 and 5 are X53-II plasmid samples that are not digested. Lanes 4 and 6 are X53-II plasmid samples that are digested with EcoRI. Lane 7 is an undigested plasmid, pBR322. Lane 8 is a plasmid pBR322 digested with EcoRI. Lanes 7 and 8 serve as a positive control.

66

3.6 Phage Sensitivity Testing:

Coliphages such as Qβ and M13 are known to infect E.coli strains carrying conjugative

plasmids (Long et al., 2005; Sobsey et al., 1995). The nine isolates X3-I, X9-I, X11-I, X16-I,

X41-I, X52-I, X53-I, X58-I and X66-I that showed the presence of plasmids were tested

for the presence of conjugative plasmids using coliphages. The Qβ and M13 phages

infect through conjugation pilus formed by the bacteria, so if the bacteria are infected,

they have conjugative genes either on the plasmid or genomic DNA. Based on this

concept, the nine isolates were tested for the sensitivity to Qβ and M13 phages. The

natural host for Qβ and M13 phages are E.coli. Three E.coli isolates X3-I, X52-I and X58-I

that showed the plasmid presence were tested for conjugative plasmid, but none of

them were infected by the coliphages (Figure 14). We also tested other isolates (non E.

coli) that showed plasmid presence even though they are not the natural host for these

phages. As expected, none of them were infected (data not shown here).

There could be two reasons for the isolates not being infected. First, all these

plasmids might not be conjugative; i.e. these isolates were not able to form conjugation

pilus. Second, even if the plasmids were conjugative, the conjugative function was

repressed for some reason. It has been shown that conjugative plasmids may carry

fertility inhibition factor or fin genes which are able to suppress the formation of

conjugation pilus in order to prevent infection caused by bacteriophages (Haft et al.,

2009). Conjugation adds stress to the host bacteria and these bacteria tend to grow

relatively slowly compared to the one without conjugation factors. The fertility

inhibition helps the host cells to multiply rapidly and dominate the population, so the fin

67

genes are turned on to reduce the cost to the host bacterium (Haft et al., 2009; Mc

Ginty and Rankin, 2012). Therefore, it could be possible that the E.coli isolates we tested

might have carried fin genes which prevented them from being infected by Qβ and M13

phages.

Figure 14: Phage sensitivity testing. On the left is a positive control, E.coli XK1502 infected with two phages: M13 and Qβ. The visible clearing or plaque formation on the lawn of bacteria represents the sensitivity of this bacteria to the phages. On the right is a negative control, E.coli MC4100 infected with the same phages but no plaques are produced. At the bottom is an isolate E.coli X52-I infected with the same phages. The absence of plaque formation indicates this isolate was not able to form conjugation pilus.

68

3.7 Tripartite Mating:

Tripartite mating was performed to identify mobilizable plasmids. This mating

process requires three strains: a donor, a helper and a recipient. The donor may carry

mobilizable plasmid but is incapable of forming conjugation pilus because of the

absence of tra gene function. The helper will provide the missing tra function to the

donor, so the donor will form conjugation pilus and transfer their gene to the recipient

(Droge et al., 2000; Schluter et al., 2007). By this assay, mobilizable plasmids can be

identified.

As mentioned in a previous section, the isolate E.coli X3-I was tested for the presence

of conjugative plasmid using the phage sensitivity assay, but it was negative for

conjugative plasmid. Before tripartite mating, the isolate X3-I was plated with the

recipient to reconfirm the absence of conjugative plasmid (data not shown here). After

reconfirmation, tripartite mating was performed with the isolate (X3-I) to identify the

presence of mobilizable plasmid. The donor was resistant to 3 drugs (ampicillin, rifampin

and chloramphenicol), the helper was nalidixic acid resistant, and the recipient was

resistant to streptomycin (Figure 15a and 15b). The antibiotic plates were made in all

different combinations of these 5 drugs, and each plate was divided into four sections:

(1) donor alone, (2) recipient alone, (3) helper alone, and (4) recipient + donor + helper.

Section 4 represents tripartite mating in which the donor isolate E.coli X3-I is plated

together with the helper and recipient E.coli on all different antibiotic plates (Figure 15a

and 15b). If the donor (X3-I) carried mobilizable plasmid, it will transfer the resistance

plasmid to the recipient and form colonies on the antibiotic plates. Unfortunately, this

69

isolate did not form a transconjugant or bacterial colony on any of the antibiotic plates,

so the donor lacks both the conjugative and mobilizable plasmids (Figure 15a and 15b).

We tested only this isolate (X3-I) because it has a resistance marker different from the

helper and recipient. We are not able to test other isolates because they are multi-drug

resistant and carried the same resistance marker like the helper and recipient. If we test

these isolates using the same procedure, then we would not be able to distinguish

between the donor, helper and recipient. We had a trouble finding a strain that has a

resistance marker different from the donor. Many of the strains available in CGSC and

ATCC bacterial strain banks were not helpful because they carried the same resistance

marker as the donor. We did not test the other isolates because of these complications.

70

Figure 15a: Resistance profile of the donor, helper and recipient strains. The donor isolate X3-I is resistant to ampicillin, rifampin and chloramphenicol. The helper is resistant to nalidixic acid and the recipient is resistant to streptomycin. These antibiotics plates serve as a positive control for tripartite mating process and confirm the resistance pattern of the strains.

Figure 15b: Tripartite mating process. As mentioned in Fig 9a, the donor was resistant to 3 drugs, and both helper and recipient were resistant to one drug, so antibiotic plates were made in all different combinations of these 5 drugs. Each plate was divided into four sections: (1) donor alone, (2) recipient alone, (3) helper alone, and (4) recipient + donor + helper. Section 4 represents tripartite mating in which the donor isolate E.coli X3-I is plated together with the helper and recipient E.coli on all different antibiotic plates. None of them resulted in colony formation, so this isolate is neither conjugative nor mobilizable type.

71

4. Conclusions:

Biosolids are used as fertilizer in agricultural crops and home gardens all over the

United States, but there is great concern regarding their application. It has been shown

that antibiotics persist in the environment for a long time and reach ground water or

aquatic sediments through application of liquid manure or sewage sludge as fertilizers

(Kuhne et al., 2001; Kummerer, 2001). Antibiotics present in sewage select resistant

bacteria, and there is increased risk of spreading drug resistance genes among members

of bacterial community. Previous studies have shown that application of manure as

fertilizer is unsafe because it can act as a reservoir for pathogens carrying drug

resistance plasmids (Binh et al., 2008). In addition, viruses present in sewage are

maintained because they are more resistant to ammonia and heat treatments (Lund et

al., 1996). These studies show that biosolids contain resistant bacteria and viruses that

can transfer their resistance genes to other bacteria. Currently, biosoilds are tested only

for the indicator organisms such as fecal coliforms, but it is better to test the biosolids

for the presence of drug resistant bacteria, human viruses and bacteriophages. Such

studies should be carried out to avoid the risks of contaminating garden and agricultural

soils with pathogens and to reduce infections in humans and animals. Alternately, the

biosolids should be sterilized before being sold as fertilizer or soil conditioner.

The purpose of my study was to determine the presence of drug resistant bacteria

and resistance (R) plasmids in Cheney biosolids. We identified bacteria (68%) were

resistant to two or more drugs tested and many of these resistant bacteria (13.2%)

72

carried resistance plasmids. The R-plasmids were able to transfer successfully to a

laboratory strain of E.coli. The resistant bacteria (13.2%) were tested for the presence of

conjugative plasmid using Qβ and M13 phages, but none of the bacteria were

conjugative type. In addition, resistant bacteria (13.2%) were tested for the presence of

mobilizable plasmid by tripartite mating, but none of the bacteria were mobilizable type.

As a result, the resistant bacteria tested were neither conjugative nor mobilizable type.

One of the obstacles for conjugation and tripartite mating is the availability of a

suitable recipient bacteria with unique resistance marker. This problem can be solved by

developing a unique recipient using recombinant DNA techniques. The presence of

conjugative and mobilizable plasmids in biosolids can be further analysed by relaxase

screening method (Alvarado et al., 2012). The biosolids should be tested further for the

presence of enteric viruses of health concern either by monitoring the presence of

bacteriophages using PCR techniques (Fout et al., 2003; Sobsey et al., 1995). In

conclusion, Cheney biosolids do contain drug resistant bacteria despite of all the waste

water treatment processes, so it is better to enhance treatment regulations to avoid

serious health effects.

73

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81

VITA

Author:

Gaayathiri Paramasivam

Place of Birth:

Kanchipuram, Tamil Nadu, India

Education:

Masters of Science, Biology

Eastern Washington University (EWU) – Cheney, WA 2011 – 2013

Masters of Science, Biotechnology

Annamalai University – Chidambaram, Tamil Nadu, India

2002-2007

Award:

Swartz-Narrance Graduate Scholarship, Eastern Washington University, 2011 - 2012

Research Experience:

Graduate Research, Department of Biology, Eastern Washington University, Cheney, WA. Winter 2011– Winter 2013, PI: Dr. Prakash Bhuta

Detection and Characterization of Antibiotic Resistance Plasmids in Cheney Biosolids

Graduate Research, Department of Biotechnology, Annamalai University, Chidambaram, TN, India. Fall 2002– Spring 2007, PI:Dr. K. Sathiyamurthy

82

Identification and Characterization of Bacteria from Various Environments

Teaching Experience:

Eastern Washington University, Spring and Summer 2012

Molecular Biotechnology (Biology 489)

Microbiology (Biology 301)

Community Involvement:

Washington Science Olympiad, Cheney, WA March and April 2012

Society for applied Microbiology - student member 2011-current

References:

Dr. Prakash Bhuta, Department of Biology, EWU. pbhuta@ewu.edu

Dr. Andrea Castillo, Department of Biology, EWU. acastillo@ewu.edu

Dr. LaVona Reeves, Department of English, EWU. drllreeves@yahoo.com

Christopher Ryan, Department of English, EWU. cjhrise@live.com